Immobilized buffered gels and membranes of hydroxy groups containing polymers

ABSTRACT

A gradient mixer for the preparation of immobilized pH-gradient (IPG) gels, which includes a positive displacement syringe dispensing device (4,5) driven by a controlled speed motor (1); a mixer driven by a magnetic stirrer (6); a temperature controlled circulating air oven for the mixer/dispensing syringes (7); a cassette into which mixed solutions of acidic/basic gels are dispensed and the IPG gel is shaped (9); and a second temperature controlled circulating air oven for the cassette (8).

This is a divisional application of Ser. No. 08/067,617, filed May 27,1993, now U.S. Pat. No. 5,430,099

The present invention relates to immobilized buffered gels and their usein electrophoretic processes, particularly in preparative isoelectricfocusing (PIEF) for separating (high molecular) organic compounds, suchas proteins or peptides from biological mixtures, and in immobilizedpH-gradients for isoelectric focusing slabs and columns.

More particularly, the present invention is directed to new polymericreaction products and methods for their preparation, and their use forpreparing said immobilized buffered gels of for example a fixed pH-valueor of a pH-gradient. These gels are suitable media for preparingmembranes, plates or columns, such as plates or columns of immobilizedpH-gradient gels (IPG-gels) which can be used e.g. in capillaryelectrophoresis (CE).

Biologically active materials require high purity to prevent deleteriousside effects by impurities, which are often also biologically active.This is especially true, and particularly difficult to achieve, forproteins made by biotechnological processes, such as fermentations,because they may contain other proteins (as impurities) separated byeach other by as little as 0.001 pI units (pI=isoelectric point).

(Preparative) isoelectric focusing is one of the few practical(electrophoretic) methods for achieving protein purification for suchclosely matched molecules. It is certainly the most economical methodwhen it can be made to work.

A continuously working PIEF-device is for example disclosed in EP-A-287513, using polyacrylamide gel membranes. These membranes are, however,toxic because of their carcinogenic monomer (acrylamide), and attemptsfor making nontoxic gel(membranes) from, for example, agarose were nothighly successful (P. Wenger et al., J. of Biochemical and BiophysicalMethods 14, 29-43 (1987)). The polyacrylamide gels are also relativeweak mechanically.

Polyacrylamide gels are further limited in PIEF-processes by aconsiderable sieving effect which does not allow free flow of proteinmolecules with molecular weights greater than 500 kDa (kilo-Dalton).This prevents the efficient separation of high molecular weight proteinsor viruses in the preparative or analytical devices (gel membranes,IPG-plates or columns).

Derivatized agarose and its utilisation for gel electrophoresis aredisclosed in U.S. Pat. No. 4,319,975 to Cook. The agarose derivatives,among them those of non-ionic triazines, show a decrease in pore sizewhich is useful for sieving properties. These derivatives are useful forcontinually moving molecules, but are not applicable to isoelectricfocusing. Further, the Cook reference does not teach the production orthe application of immobilized buffering groups on polymers and theirformation into PIEF or IPG gels.

While the polyacrylamide gels are not entirely satisfactory as PIEF gelsand the non-ionic agarose derivatives cannot be used as such, thesedisadvantages can be overcome by the inventive gels which arenon-sieving gels for free passage of all molecules and contain acid andbasic groups together in the right proportions to give bufferingcapacity, at a fixed pH-value. Other advantages of the inventive gelsare their low toxicity and better pH-stability.

Therefore, it is one object of the present invention to provide newreaction products of a hydroxy group containing neutral polymer with ananionic (acidic) or cationic (basic) diazine or triazine derivative.

Other objects of the present invention are the process of thepreparation of said reaction products, an immobilized buffered gel whichcomprises an optionally cross-linked matrix of a mixture of said acidand basic reaction products, a process for the preparation of theimmobilized buffered gels, the formation of the gels into membranes orimmobilized pH-gradient gel plates or columns, the gradient mixer toprepare these membranes, plates and columns,as well as their use inpreparative isoelectric focusing electrophoresis, e.g. for separatingbiologically active components from biological mixtures.

These and other objects of the present invention will become apparentfrom the following detailed description.

The inventive reaction products are preferably those wherein the hydroxygroup containing neutral polymer is a natural or a synthetic homo- orcopolymer and the diazine or triazine derivative is

a reactive acid reaction product of (a) an amino-carboxylic oramino-sulfonic acid and (b) a di- or triazine containing a nucleophlicleaving group, or

a reactive basic reaction product of (c) a mono- or polyamine and (b) adi- or triazine containing a nucleophilic leaving group.

Suitable, natural hydroxy group containing polymers are e.g. thepolysaccharides, preferably agarose.

The synthetic homo- or copolymers can be selected from the vast numberof hydroxy group containing polymeric species; preferred are thepolyvinyl alcohols or the copolymers on the basis of vinylalcohol;further a poly-(hydroxyalkyl)- or a poly-(hydroxyalkoxyalkyl)-acrylateor-methacrylate, a homopolymer based on anN-acryloyl(methacryloyl)-tris-(hydroxyalkyl)aminomethane, or a copolymerof a hydroxyalkylacrylate or -methacrylate or a hydroxyalkoxyalkylacrylate or -methacrylate and at least one comonomer, or a block co- orterpolymer of said (meth)acrylates and at least one of said comonomer.

In these compounds alkyl and alkoxy each independently represent loweralkyl or alkoxy radicals with e.g. 1 to 5, preferably 1 to 3 carbonatoms, such as methyl, ethyl, propyl, methoxy, ethoxy or propoxy.

Suitable copolymers on the basis of vinylalcohol are those containing ascomonomers e.g. ethylene, methyl(meth)acrylate, N-vinylpyrrolidone, orpreferably a hydroxyalkyl acrylate or methacrylate with alkyl of 1 to 3carbon atoms.

Suitable comonomers that can be copolymerized with thehydroxyalkyl/hydroxyalkoxyalkyl-acrylates or methacrylates are e.g.vinyl ethers, such as vinylethylether; vinyl esters, such as vinylacetate; N-vinyl pyrrolidone; a mono- or poly-(alkoxy)-alkyl acrylate ormethacrylate, such as methoxyethyl methacrylate, methoxyethoxyethylmethacrylate, or methoxydiethoxyethyl methacrylate, or an alkylene (C₂-C₄)glycol di-acrylate or-methacrylate.

Suitable and preferred homopolymers are those based on hydroxyethylmethacrylate, hydroxyethoxyethyl methacrylate or hydroxydiethoxyethylmethacrylate, or a homopolymer based onN-acryloyl-tris-(hydroxymethyl)-aminomethane(NAT).

The reactive acid reaction product (which will be reacted with thepolymer) of(a) an amino-carboxylic or amino-sulfonic acid and (b) a di-or triazine containing a nucleophilic leaving group comprises as (a) analkylamino carboxylic or alkylamino sulfonic acid, each independentlycontaining alkyl of preferably 1 to 6 carbon atoms. Examples are: aminoglycolic acid, glycine, 3-amino propanoic acid, 4-amino butyric acid, or2-amino propane sulfonic acid.

The di- and triazines (b) contain at least two nucleophilic leavinggroups (groups that are split off when the component undergoes anucleophilic substitution reaction) which are first of all halogenatoms, such as bromo, but preferably fluoro and chloro; furtherammonium, such as tri(lower)alkyl ammonium or (optionally substituted)pyridinium; sulfo or sulfonium (R₃ S⁺ -, R=C₁ -C₄ -alkyl); andphosphonium.

Representative examples of the di- and triazines are:

(A) s-triazines containing at least two reactive identical or differenthalogen atoms bound to carbon atoms, for example cyanuric chloride,cyanuric fluoride, cyanuric bromide and also primary condensationproducts of these cyanuric halides and, for example, water, ammonia,amines, alkanols, alkylmercaptans, phenols or thiophenols; furtherphenols, anilines, alkanols and alkylamines containing ionic groupswhich will render the dihalogenated triazines water-soluble. Such ionicgroups are sulfonic, carboxylic, quaternary ammonium, sulfonium orphosphonium groups;

(B) pyrimidines containing at least two reactive identical or differenthalogen atoms, such as 2,4,5-trichloro/trifluoro/tribromo-pyrimidines,which can be further substituted in 6-position, for example by alkyl,alkenyl, phenyl, carboxyl, cyano, nitro, chloromethyl, chlorovinyl,carbalkoxy, carboxymethyl, alkylsulfonyl, carboxamido or sulfamido, butfreferably by halogen (fluoro, chloro, bromo). Particularly suitablehalogeno pyrimidines are 2,4,5-trichloro-, 2,4-difluoro-5-chloro- or2,4,5,6-tetrachloro-pyrimidine; further water-soluble derivatives ofpyrimidine similar to those of (A), above, which do not contain acid orbasic groups, which are not replaceable and which contribute to the pH.

(C) halogenopyrimidine-carboxylic acid halides, for exampledichloropyrimidine-5- or -6-carboxylic acid chloride;

(D) 2,3-dihalogeno-quinoxaline- or-phthalazine-carboxylic acid halidesor-sulfonic acid halides, such as 2,3-dichloro-quinoxaline-6-carboxylicacid chloride or bromide;

(E) halogeno-6-pyridazonyl-1-alkanoyl halides or-benzoyl halides, forexample 4,5-dichloro-6-pyridazonl-1-propionyl chloride or-1-benzoylchloride.

The preferred compounds are s-triazines and pyrimidines substituted byactive leaving groups, such as halogen atoms, prefearably fluoro andchloro, quternary ammonium groups or sulfo (--SO₃ H).

The reactive basic reaction product (which will be reacted with thepolymer) of (c) a mono- or polyamine and (b) a di- or triazinecontaining a nucleophilic leaving group comprises as (c) an alkylmono-or an alkylene polyamine of 4 to 10 carbon atoms. Representativeexamples of (c) are 2-aminoethyl-morpholine, 3-aminopropyl-morpholine,2-(dimethylamino)-ethylamine, 3-(dimethylamino)-propylamine,3-(diethylamino)-propylamine, and 2-(triethylammonium)-ethylaminechloride. Component (b) is as defined above.

Examples of the reactive acid reaction products (which impart buffercapacity to the polymers (gels)) are: 1-(amino-2-propane sulfonicacid)-3,5-dichlorotriazine, 1-(amino glycolicacid)-3,5-dichlorotriazine, 1-(glycino)-3,5-dichlorotriazine, 1-(aminopropanoic acid)-3,5-dichlorotriazine, 1-(amino-butyricacid)-3,5-dichlorotriazine. Examples of the reactive basic reactionproducts which also are used to impart buffer capacity to the thepolymers are: 1-(2-morpholino-ethylamino)-3,5-dichlorotriazine,1-(3-morpholino-propylamino)-3,5-dichlorotriazine,1-(2-dimethylamino-ethylamino)-3,5-dichlorotriazine,1-(3-dimethylamino-propylamino )-3,5-dichlorotriazine,1-(3-diethylamino-propylamino )-3,5-dichlorotriazine and1-(2-triethylammonium-ethylamino)-3,5-dichlorotriazine chloride.

The reactive acid and basic reaction products can be prepared byreacting (a) or (c) with (b) according to known chemical processes. Theacid or basic moiety does not react with (b) under the conditionsemployed. The acid groups are preferably chosen from carboxylic acids,and the amino groups are chosen from tertiary alkyl or heterocyclicamines. These amines should not react under the conditions of use withthe other reactive groups of (b) (or of different reagents which may beused to cross-link the polymer (gel)), and this generally means that thealkyl side chain should contain more than one carbon atom in at leasttwo of the alkyl radicals (which prevents reactivity of tertiary amineswith halo triazines or diazines because of steric hindrance, and theheterocyclic amines should contain sterically arranged groups whichprevent reactions).

Where the heterocyclic amines can react with component (b) to formquaternary ammoniums, they should react much slower than the designedbinding groups, and if they react at other conditions after theacid/basic reaction products (buffering reagents) are bound, they shouldform reactive groups which can be displaced by the functional group ofthe polymer (gel) to bind the buffering reagents to the polymers (gels).

The acid and basic diazine and triazine derivatives as hereinbeforedescribed (which serve as pH-determining moieties) are reacted with ahydroxy group containing neutral polymer; the resulting acid and basicpolymeric reaction products are mixed together (titrated against eachother) to form a solution of the desired pH-value, set in a mold andthen gelled, e.g. by lowering the temperature (when agarose derivativesare used) or by chemically cross-linking. These immobilized bufferedgels constitute a further object of the present invention.

The inventive gels can be prepared by various processes, one of whichcomprises reacting simultaneously the acid and the basic diazine ortriazine derivatives with the hydroxy group containing neutral polymerin a mold and gelling them.

Alternate routes to the inventive immobilized buffered gels comprise

(1) reacting separately the acid diazine or triazine derivative and thebasic diazine or triazine derivative each with the hydroxy groupcontaining neutral polymer,

(2) titrating the polymeric reaction products of (1) batchwise againsteach other to form a polymeric mixture of a predetermined singlepH-value, or in a continuous manner to form a polymeric mixture of apH-gradient, and

(3) casting the polymeric mixtures of (2) into a mold and gelling them;or

(1) reacting a reactive diazine or triazine, non-derivatized with anacid or basic pH-determining compound, with the hydroxy group containingneutral polymer,

(2) reacting the polymeric reaction product of (1) separately with anacid and an basic pH-determining compound,

(3) titrating the polymeric reaction products of (2) batchwise againsteach other to form a polymeric mixture of a predetermined singlepH-value, or in a continuous manner to form a polymeric mixture of apH-gradient, and

(4) casting the polymeric mixtures of (3) into a mold and gelling them.

The preparation of the derivatized polymers (gels) may thus takedifferent pathways: In one approach, the triazine or diazine derivativesof the acid or basic buffer (compound with a pH-determining moiety) areprepared and then reacted with the polymer. In this case, the reactivereagent may be prepared in preferably non-aqueous solutions or inaqueous ones when one of the components is not soluble. The derivativeis then reacted with the gel at a pH and temperature which is allowed bythe second halogen atom on the triazine and which leaves the thirdhalogen atom unreacted. In the case of triazines, the cyanuric chlorideis derivatized with the acid or basic buffering groups at 0° to 10° C.,and reacted with the gel at 15° to 25° C. Thus if, when working inaqueous solutions, the second chloro atom hydrolyzes to hydroxy, thisderivative has only the third chloro atom, which is not reactive andwill not react with the gel and will be washed out in the purificationstep.

Thus, after the derivatization of agarose or other polymers, theobtained polymeric products are purified. Being not cross-linked theyare redissolved and titrated against each other to the proper pH-valueand then formed into the desired configuration, and geller and/orcrosslinked as required.

In an other approach the polymer may be first derivatized through itshydroxy groups with the underivatized halogeno di- or triazine(tetrachloro pyrimidine or preferably cyanuric chloride), the excessreagent washed away, and then reacted with the acid or basic compound.These compounds may be the said amino acids (e.g.4-aminobutyric acid)for the acid derivatives, and primary alkyl tertiary amines (e.g.2-aminoethyl-morpholine) for the basic derivatives. They may also be,but less preferred, hydroxy acids or hydroxy tertiary amino derivatives.All the steps may be carried out in solution, but it is alsoadvantageous to carry out the reactions on the polymer (gel).

It is further advantageous to work in non-aqueous systems wheneverpossible to avoid competitive hydrolysis of the active halogen atoms ofthe di- or triazines. Thus, for example, in the case of agarose, the gelis made from water solutions by lowering the temperature of the sol toget a gel, replacing the water with acetone, and reacting with cyanuricchloride in acetone with a non-nucleophilic acid acceptor, such asN,N-dimethyl aniline, diisopropyl amine or diisopropylethyl amine, at 0°to 5° C. After a period of time, the gel is washed off unreactedcyanuric chloride with acetone, then reacted with an excess of the acidor basic reagent to introduce the buffering groups, and the excessreagent washed out. This approach works well with the basic bufferingreagents, as they dissolve well in non-aqueous solutions like acetone ortetrahydrofurane. The acid derivatives do not dissolve so well innon-aqueous solvents and must be used in general in aqueous mixtures orwith bulky aliphatic or aromatic counter ions to the acid groups toincrease solubility in solvents. An aqueous mixture that can be used ise.g. acetone/water (90/10).

Alternatively, the water of the gel can be replaced by a solvent likeacetone, and the acetone swollen gel can be reacted with the triazine ordiazine derivative in aprotic solvents, such as acetone, in order tobind the buffer groups to the gel. The gel is washed and then remeltedand titrated to get the desired pH and buffer capacity.

In both, the polymerization to a gel and the cross-linking of anexisting polymer, the relationship between such parameters as the degreeof cross-linking, crystallinity, hydrophilicity and permeability areimportant. The gels (hydrogels) which do not innately form open networkstructures as e.g. agarose must be made into a sufficient porousstructure, which is, nevertheless, mechanically strong.

A preferred way to form such a gel and a gel membrane would be asfollows:

A solution of the (water-soluble) polymers are allowed to react, and asolvent swollen gel is formed when the polymer is cross-linked.Pore-forming agents (e.g. leachable polymers, cosolvents) may be used toget large pores. For example, the permeability of poly-(2-hydroxyethylmethacrylate) membranes (J. Biotaed. Mat. Res. 15, 307, 1981) have beenstudied, and it has been found that the structure of themembrane-forming gel depends on the amount of the cross-linking agent.At low concentrations of the cross-linking agent, the transport of waterand solutes is possible through the pores present. At highconcentrations of the cross-linking agent, the membranes are dense andblock solute passage. In the case of polyvinyl alcohol (PVA),permeability is affected by the extent of crystallinity versus amorphouscontent, which can be controlled by choice of comonomers.

Other polymers, such as those based on NAT, form porous structuresgreater than polyacrylamides, even though they are formed in the sameway of polymerization of the monomer and cross-linking duringpolymerization.

Agarose, a thermally reversible gel, on the other hand, forms gels withlarge pores on its own when cooled from a sol which are suitable forprotein permeation.

In working with thermally reversible gels, such as agarose, the best wayto form gel membranes is to first derivatize separately the agarose withthe acid and basic di- or triazine derivative and then to isolate (forpurification). The (polymeric) derivatives are then titrated againsteach other to the desired pH-range (single pH-point) for single pHmembranes or continuously to form IPG at elevated temperature while inthe sol state. To make a PIEF-membrane, the sol titrated to the requiredpH is cast in a mold and then left to cool and gel.

The reason for derivatizing the polymer prior to casting is that controlover the pH or pH-gradient is is more accurately carried out bytitration of preformed species in solution, than on gels already formedinto the shaps they will be used.

For making IPG-gels in plate or column or capillary form, one agarosederivative in sol form is titrated continuously with one or more otheragarose derivatives, and added to a mold while continuously titrating toget a continuous pH gradient. The sol is then left in the mold to geland gives an IPG. In making the pH gradient, density gradients may beset up with gycerol or other suitable materials. Devices for continuousgradient making are well known in the state of the art. In working withsols and polymer solutions, it may not be necsessary to work with thesaid density gradient materials.

The inventive agarose derivatives are thermally reversible gels whichare solid at room temperature and need no cross-linking. Othernon-thermally reversible gel polymer derivatives (e.g. derivatizedpolyvinyl alcohol or poly-(hydroxyalkylmethacrylates)) must becross-linked after they are formed in the desired configuration.

If the gel membrane is made from a thermally reversible gel, as would bethe case with agarose, or from a water-swellable but not water-solublepolymer, then the gel does not have to be chemically cross-linked,although ist may be desirable to enhance extreme pH and chemicalstability.

Room temperature soluble hydrophilic polymers, such as polyvinylalcohol, should be cross-linked to prevent dissolution. Thecross-linking may be done with a cross-linker which is the acid/basicdi- and triazine derivative mentioned (buffering reagent--BR), when itcontains two or more reactive (leaving) groups towards the functionalgroups (OH-groups) of the polymer (gel), with a cross-linker other thanthe BR, or with a combination of both.

In one preferred cross-linking mode, the BR does both the cross-linkingand introduces the buffering moieties. The cross-linking should occurafter the sol or polymer solutions have been titrated into the right pHrange and formed as gels into the desired configuration (plates, columnsor capillaries).

Preferred examples of BR are dichloro triazine derivatives of tertiaryamines and alkyl carboxylic acids as mentioned hereinbefore.

Cross-linkers which only cross-link but do not buffer may be chosen fromthe list below (e.g. non-ionic triazine derivatives).

If non-thermally reversible polymers are used, then they are preferablyderivatized first, purified and then mixed in the correct proportion bytitration as required to achieve the correct pH or pH gradient, asdescribed above for agarose. They are then placed in a mold, where theyare subsequently cross-linked to maintain their shape and preserve thepH or pH gradient.

The cross-linking agents in this context are multi-functional reagentswith groups that react with hydroxy groups and do not introduce ionic,acid or basic, groups. These include aldehydes, such as acetaldehyde orglutardialdehyde; diacids, such as maleic or oxalic acid; di-esters;di-isocyanates, such as C₂ -C₄ -alkylene di-isocyanates, e.g. ethylenedi-isocyanate; epoxides (water-soluble); di-vinyl sulfones; free oretherified N-methylol ureas or N-methylol melamines, such asN,N-dimethylolurea, N,N-dimethylolurea dimethyl ether ortrimethylolmelamine dimetyl ether; eerie redox systems; and mostpreferably diazines and triazines; of these latter onestetrachloropyrimidine and in particular cyanuric chloride have provenespecially advantageous.

In the approach to forming hydrogels from existing polymers, BR will bebound to the hydrophilic polymers prior to cross-linking to form the gelor be carded out during the cross-linking step. For example, BR withhalogen di- or triazinyl groups may be prepared and then reacted withthe pendants of the hydrogel polymers.

Good candidate polymers for this approach are hydrogels, such asagarose, polymers based on NAT and PVA. The BR may also be used withhydrogel monomers during polymerization to form cross-linked hydrogels.One benefit of this approach is improved pH stability over the ester oramide type BR. All BR achieve the maximum buffer capacity for the chosenisoelectric point. The criteria for maximizing buffer capacity is wellknown, and one preferred approach is to use appropriate mixtures of weakacid and basic buffers wherein the difference between their respectivepK-value is less than 2.

The modes of reacting of the BR with the hydroxy groups of the polymers(gels) follow the basic chemistry of di- and triazines. Their reactivitycan be enhanced under basic conditions. The components can be mixed andformed into a polymer solution by the followiong different modes:

(1) The components of polymer and BR are mixed at a pH at which they donot react rapidly and after forming a homogeneous solution anddegassing, the solution's pH is made more basic to react the polymer,and the polymer is cast into a mold or on a substrate, where it isallowed to gel by either cross-linking and/or cooling. A gel with boundBR moieties is obtained.

(2) The same procedure as in (1) is followed except that after the pH isadjusted to react the BR, the solution is continued to stir prior todegassing and casting; then casting and adjusting the pH to getcross-linking reactions to gel formation.

(3) The solution's pH may be adjusted at the outset of mixing thecomponents and the remainder of the procedures described in (1) and (2)may be followed.

(4) Since both acid and basic BR must be bound to the gels, they may bebound either together in the same polymer solution or in separatepolymer solutions, and then, after reaction, the separate polymersolutions may be mixed. The detailed sequence for each solution and/orfor the combined polymer solution may be as described in (1) to (3),above, or a combination thereof.

(5) For thermal reversible gels, it is preferable to carry out thebinding reaction at the lowest temperature at which the gel is insolution, though higher temperatures may be used. The temperature atwhich the gel is cast into the mold may be the same at which the bindingreaction occurs or it may be a higher temperature where the gel is lessviscous and may be poured and degasseal to form a more uniform gel. Thegel may be kept at this elevated temperature for some time, whereadditional binding of the BR may ooccur if it was not completed at thelower temperature.

It is advantageous to carry out the binding reaction at lowertemperatures to minimize the hydrolysis rate of the BR, in comparison tomaximizing the binding of BR to the polymer. On the other hand, highertemperature may be optimum for casting and degassing because of thereduced viscosity. Alternatively, the BR may be bound to the gel, washedof excess reagent, and then melted, titrated one with the other, andcast into the buffered gel.

For water-soluble polymers which are not thermally reversible gels, theabove heating protocols may be followed, or both the binding of the BRand the casting of the derivatized polymer may be carried out at thesame temperature. For water-soluble gels, the binding and cross-linkingreactions may occur at temperatures of from -10° to 100° C., butpreferably between 4° to 90° C. Thermally reversible gels like agarosemay be reacted with the BR at 40° to 90° C. and preferably 40° to 60° C.(in order to prevent competitive reactions (hydrolysis) the temperatureshould be kept--whenever possible--to the minimum in which the polymersolution is still a solution and not a gel), and then raised to 70° to95° C., preferably 90° C. for casting.

The pH ranges for binding the reagents are, to a certain extent, afunction of the polymer stability, and where the BR is required to onlybind to the polymer or also to cross-link it. For agarose and otherthermally reversible gels the primary task is to bind the buffer moiety.In addition, gels made from naturally occuring polysaccharides, such asagarose, must be reacted under relatively mild pH conditions.

Thus for agarose the dichlorotriazine buffers are reacted at pH valuesof from about 6.0 to 8.0. Water-soluble and pH stable polymers, such aspolyvinyl alcohol, are reacted, in the pH range of 6.0 to 11.0. The morebasic the pH range (e.g. 8.0 to 11.0) that can be used for reacting, theless reactive moieties on the BR are necessary to achieve cross-linking;as for example the third chloro atom of the chloro triazine moleculeafter two chloro atoms have reacted with the buffer group and onehydroxy group of the polymer (gel), repectively.

When starting from monomers a (co)polymerization step is involved in thesequence of reaction steps that lead to the inventive immobilizedbuffered gels. These methods of preparation comprise e.g.

(1) reacting separately the acid diazine or triazine derivative and thebasic diazine or triazine derivative each with a hydroxy groupcontaining neutral polymerizable monomer,

(2) titrating the polymerizable compounds of (1) batchwise against eachother,

(3) casting the mixture of the polymerizable compounds of (2) into amold,

(4) polymerizing the mixture of (3) in the presence of a cross-linker,and optionally together with a comonomer, and

(5) gelling the polymeric reaction products of (4); or

(1) reacting separately the acid diazine or triazine derivative and thebasic diazine or triazine derivative each with a hydroxy groupcontaining neutral polymerizable monomer,

(2) separately polymerizing the reaction products of (1), optionallytogether with a comonomer,

(3) titrating the polymeric reaction products of (2) batchwise againsteach other to form a polymeric mixture of a predetermined singlepH-value, or in a continuous manner to form a polymeric mixture of apH-gradient, and

(4) casting the polymeric mixtures of (3) into a mold and gelling themin the presence of a cross-linker.

Copolymerization/cross-linking reactions to produce polymer gels,especially poly-(hydroxyalkyl methacrylates), are known from the stateof the art. Initiators that can be used in these reactions include bothradical and anionic initiators. Azo-bis-isobutyronitrile (AIBN) iswidely used, although there are advantages of the use ofazo-bis-methylisobutyrate and other similar chemicals. Peroxides, suchas benzoyl or cumyl peroxide, or peracids can be used as well.

Solvents may be added during the polymerization to decrease theviscosity of the solution.

Chain transfer is a typical problem in these reactions. For example,during the polymerization of 2-hydroxyethyl methacrylate in the absenceof a cross-linking agent, chain transfer to the polymer may be observed,which leads to the formation of cross-linked poly-(2-hydroxyethylmethacrylate)(PHEMA).

Amongst other properties, the copolymerization of hydrophilic andhydrophobic monomers also allows the control of hydrophilicity. Therelatively high hydrophobicity of the usual chemical polymerizablecross-linking agents also affects the overall hydrophilicity of thehydrogel formed, which may, of course, be raised by exchanging thecross-linking agent for a more hydrophilic compound. It must beremembered that, with hydrogels formed by chemical cross-linking, thecopolymers obtained depend on the constraints of the copolymerizationsystem, especially with respect to the possible inhomogeneity ofcomposition of the copolymer formed, particularly if thecopolymerization parameters differ from each other.

The chemical character of hydrophilic groups differs not only inaffinity to water but also, equally important, in polarity. This makesthe character of some gels either completely neutral, e.g. PHEMA, orpolymers of glucose and sucrose methacrylates--or capable ofionization--e.g. polyacrylic and polymethacrylic acids and coplymers onthe basis of (meth)acrylic acid--or basic, such as polyvinyl pyridineand its copolymers and derivatives. negatively and positively chargedpolymers mixed together can form water-insoluble, but swelling,complexes which behave as hydrogels. For the present invention, however,only neutral polymers which prevent adsorption and electro-osmosis areof interest. One especially neutral gel made by polymerization is thatof NAT.

One preferred way to form the immobilized buffered gels and thecorresponding membranes by polymerization would be as follows: (Aqueous)solutions of the derivatized (acid/basic) polymerizable monomers arebatchwise titrated against each other to achieve the required pH-value,e.g. for PIEF-membranes, and then cast into a configuration; or when thetitration is carried out continuously with two different solutions, themixture is added to a mold to form a pH-gradient. The gradient in thiscase may be stabilized with e.g. glycerol. After pouring into the moldthe mixtures are polymerized in the presence of polymerizationinitiators, crosslinking agents (monomers), and optionally comonomers,e.g. by applying heat; thus preserving the shape and the pH-gradient. Agel structure is formed, swollen by the solvent. The pore size may, ofcourse, be controlled by the concentration of the cross-linker (and e.g.its hydrophobicity) and the initiator (consistent with mechanicalstrength), but just as important may be pore-forming agents. Such agentsmay be leachable polymers (neutral and non-charged), or cosolvents whichprecipitate the polymers as they are formed.

In an alternate route the acid/basic di- or triazine derivatives(buffering reagents--BR-) may react with the polymerizable monomer orthe (formed) polymer in the process of polymerization. Thus, the BR maybe added at any time before or during the polymerization. In addition toBR, other materials may be added to facilitate the reaction between themand the hydroxyl groups of the monomers or polymers (matrix). Examplesof such additives are catalysts such as tertiary amines, e.g.trimethylamine, or aromatic or heterocyclic amines, such as optionallysubstituted pyridines, e.g. 4-(dimethyl)-amino pyridine, ornon-nucleophilic proton acceptors, such as diisoprpyl amine,2,6-lutidine and 2,4,6-collidine. Particularly powerful catalysts are4-N,N-dimethylamino pyridine and 4-pyrrolidino pyridine.

The hydroxy group containing polymerizable monomers reacted with BR area further object of the present invention. As indicated above, they canbe (co)polymerized in the presence of suitable initiators,cross-linkers, comonomers and optionally other additives to form, aftercasting into a configuration and gelling, the immobilized buffered gelsand membranes, respectively.

Suitable monomers are those mentioned hereinabove for the preparation ofthe hydroxy group containing polymers; useful BR are also mentioned.

The general reaction scheme to get these compounds is as follows:Polymerizable Monomer-OH+L-BR=Polymerizable Monomer-O-BR+HL(L=nucleophilic leaving group, such as chloro).

The bound acid or basic group molarity may vary from about 10 to 5000mmol, preferably from 50 to 2000 mmol, and most preferably from 150 or200 to 1500 mmol per kg of dry gel.

In the case of wet (aqueous) gels, e.g. 2% gels, the amount of saidgroups comprises only the corresponding part, e.g. the 50^(th) part. Inthe case of wet agarose gels (e.g.2% gels) the preferred range is from 1to 100(50) mmol, preferably from 1 to 20 mmol, and most preferably from3 to 15 mmol per kg of wet gel.

The inventive immobilized buffered gels, when formed e.g. in membranes,pH-gradient gel plates or columns, may be used in electrophoreticprocesses, most preferably in preparative isoelectric focusingelectrophoresis (PIEF) for separating amphoteric substances, such asbiological materials.

The principle of isoelectric focusing is based on the fact that certainbiological materials (such as proteins, peptides, nucleic acids, andviruses) are amphoteric in nature, i.e. they are positively charged inan acid medium and negatively charged in a basic medium. At a particularpH value, called the isoelectric point (pI), these biological materialswill have a zero net charge.

Being charged in a pH gradient, the biomaterials migrate under theinfluence of an electric field until they reach the ph of theirisoelectric point. At the isoelectric point (zero net charge), thebiomaterials are not influenced by the electric field. Diffusion of"focused" biomaterials away from their pI will cause them to once againbecome charged, whereby they will electrophoretically migrate back totheir pI. Thus, the biomaterials focus into narrow zones from which theycan be selectively separated.

The inventive immobilized buffered gels in membrane form can form gelbarriers that can be used e.g. in PIEF devices. The barriers arecharacterized by a constant pH, close to the pI of the targetbiomaterial (protein), which keeps this protein entrapper within a cell.At the same time, the charged impurities are driven through the gelmembranes by an applied electric field perpendicular to the membrane.

The pH of the gel membrane is chosen to be slightly higher or lower thanthe pI of the target protein (or both membranes may be at the same pHequal to that of the isoelectric point), so that when it occasionallyapproaches the membrane surface, it acquires electric charge, whichcauses the protein to be pulled back from the membrane into the centralsolution. Thus the solution containing the desired material (proteinetc.) can be circulated in the feed compartment of the PIEF device,continually removing the impurities and purfying the desired material,which is focused in said compartment. By such a method, proteins can beseparated differing by as little as 0.01 pH units in their pKi values(pK of the isoelectric points).

BRIEF DESCRIPTION OF THE INVENTION

The inventive gel membranes are designed for an electrophoreticapparatus (continuous isoelectric focusing device) genericallyrepresented in FIG. 1, and known from EP-A-287 513.

Other devices wherein the inventive gels or membranes can be used aredescribed in EP-A-323 948 and EP-A-369 945.

FIG. 2 schematically decribes the general layout of the gradient mixer.

The device of FIG. 1 basically comprises a flow chamber (8), containingthe protein feed solution connected with two containers (3) and (4),separated by the gel membrane with immobilized pH barrier (2). Themembrane close to the anode (+) may have an isoelectric point just belowto the electric point of the protein being purified, and the membraneclose to the cathode (-) may have an isoelectric point just above thatvalue.

This device uses an isoelectric focusing technique wherein the proteinof interest is not electrophoretically driven into a gel matrix, but iskept in an isoelectric state in the liquid stream, and only theimpurities are forced to focus in the gel membrane phases.

Because the electrophoretic separation is performed in a pH gradient,all the species having an isoelectric point within the pH gradient aredriven by a voltage gradient to the particular zone where they exhibitzero net charge and in which they remain stationary as long as theelectric field is applied. The protein of interest is alreadyisoelectric in the flow chamber (8) which constitutes the sample feedsystem. Therefore, said protein is not forced to flow by the electricfield.

The gel membranes may be regarded as very short pH gradients, coveringonly a very narrow pH interval. Ideally, the pH interval comprises zeropH units. Furthermore, the pH values of the membranes may have a pHvalue, which is identical to isoelectric point of the compound ofinterest. Consequently, identical membranes can be prepared instead ofmembranes differing from each other.

The currently used membranes are prepared from poyacrylamide gels, whichare operating satisfactorily except for their potentially carcinogenicleachables of acrylamide, poor mechanical strength and their sievingeffect for very large molecules (above 500 kDa). This toxicity limitstheir use for human or animal consumptions, as in pharmaceuticalapplications.

These disadvantages can be overcome by the present invention, and it isone main advantage of the the inventive gels and membranes that they arenon-toxic. Other advantages of the inventive gels and membranes aretheir fixed and constant pH and their good buffer capacity, as well astheir conductivity; they show non-sieving behaviour and allow transportof impurity macro-molecules, such as proteins and ionic polysaccharides;electro-osmotic effects can be prevented due to the minimum content ofresidual ionic groups; further, the gels and membranes eliminatehydrophobic or electrostatic adsorption of macro-molecules because theyare hydrophilic and non-charged; and finally, the gels and membranesshow particular mechanical and chemical stability.

The commonly used practice for the preparation of immobilizedpH-gradient gels (IPG) is to mix a heavy acid solution with a lightbasic solution in a predetermined and controlled manner, dispense theliquid mixture into a a specially shaped mold (cassette) an solidify themixture into a gel shaped to a sheet which is approximately 0.5 mmthick, 10 cm long and 10 to 20 cm wide.

The density of the solutions is controlled by appropriate addition ofglycerol in such a way as to generate inside the cassette a densitygradient.

The density gradient helps to maintain the shape of the pH-gradientduring the stages of the gel formation. While, as exemplified, glycerolis used to form the density gradient, the density gradient may well beformed with other substances.

The pH of the solutions is adjusted such that the acidic solution has apH equal to the basic extreme of the IPG gel.

The available pH gradient mixers are only suitable for the preparationof polyacrylamide gels in which both the acidic and basic solutions havea low viscosity and the mixing is done at ambient temperature.

These mixtures are not suitable for preparing agarose based IPG gelssince the agarose solutions are much more viscous and have to be mixedand dispensed into the cassette at the temperature of molten agarose(60° to 80° C.).

Furthermore, to achieve equilibrium for the density gradient in thecassette prior to solidification, the mixed pH gradient has to be keptat elevated temperatures (about 60° C.) for some time.

Thus, the gradient mixer (FIG. 2) for the preparation of agarose basedIPG gels--and this is a further object of the presentinvention--comprises:

a positive displacement syringe dispensing device (4,5) driven by acontrolled speed motor (1)

a mixer driven by a magnetically couple stirrer (6)

a temperature controlled circulating air oven for the mixer/dispensingsyringes (7)

a cassette into which the mixed solutions of the gels are dipensed andthe IPG gel is shaped (9)

a second temperature controlled circulating air oven for the cassette(8).

The mixer is comprised of two 15 ml glass syringes, a basic gel glasssyringe (4) and an acidic gel glass syringe (5), which are placed in aclosed circulating air oven compartment (7) preheated to the gel melttemperature, usually 70° to 80° C., by an air jet gun (10) (30 l/min,20° to 80° C.) controlled by a temperature controller and indicator(12).

The melted gel is sucked into the syringes through a three way valve(14) so that the more basic agarose gel is placed in syringe (4) and themore acidic gel is placed in syringe (5), in the procedure where theacidic syringe (5) will contain the glycerol and will have the higherdensity. If the gel is made with the basic (gel)solution being theheavier solution it will be put in syringe (5) and the acidic(gel)solution in syringe (4).

The design is such as to make sure that no air bubbles are entrapped inthe syringes or the connecting tubing.

After filling up the syringes with the respective gels, the variablespeed motor (1) (0 to 20 rpm) is started and the excess of gel is pushedout so that a predetermined volume of gel is left in each syringe. Thatvolume is determined accurately and repeatable by the location of thestart point switch (16).

In parallel, the cassette (9) (glass plate cassette, e.g. 20×10 cm) isplaced in the second circulating air oven (8) which is heated to about60° C. by the second air gun (11) (30 l/min, 20° to 80° C.) and kept atthat temperature for equilibration.

The temperature of the oven (8) is kept constant by the aid of atemperature controller and indicator(13).

When the whole system reaches equilibrium, the magnetic stirrer (6) isstarted, the three way valve (14) is rotated to a position whichconnects the two syringes (4)(5), the output flexible tube is placed inthe cassette (9) and the motor (1) is started at a speed of about 10 rpmas determined by the motor speed control (2).

As the motor rotates, a load screw (3) pushes down the syringe plungersand melted gel is dispensed from the basic syringe (4) to the cassette.At the same time acidic gel is dispensed from the acidic syringe (5) tothe basic syringe (4) gradually increasing the acidity in this syringe.If syringe (5) is the basic one (contains the basic gel solution) andsyringe (4) is the acidic one, then the situation is reversed.

Thus the gel which is dispensed to the cassette (9) will have aprogressively decreasing pH.

Since the more basic gel is also heavier (the basic syringe contains amix of agarose gel and 30% of glycerol), the gel dispensed into thecassette also has a gradually decreasing density. This is the particularcase for making an IPG with the pH range of 5 to 6 using aem- andgly-derivatives of agarose.

The density gradient imposed on the pH gradient helps to maintain thelinear shape of the pH gradient during the dispensing and solidificationstages.

There is an upper limit switch (15) and a low limit (stop point) switch(17). When the syringe plungers reach the low limit (stop point) switch(17) the motor is stopped. At that point the cassette (9) has been fullyfilled with gel.

After the gel in the cassette reaches equilibrium, the oven is shut off,cooled to ambient temperature, and the gel is released from thecassette. The gradient mixer is monitored by an electrical switcingboard (18).

The present invention is illustrated by the following examples whichshould not be considered limiting as to the inventive scope.

Parts and percentages are by weight, if not otherwise indicated.Temperatures are given in degrees Centigrade.

EXAMPLE 1

The method for preparing immobilized buffered agarose (gel) membranesfirst requires the synthesis of the buffered reagents (BR), theirbinding to the agarose polymer in separate samples for the acid andbasic BR, respectively, and mixing and casting of the sol or solution ofthe polymeric reaction products.

Preparation of acid and basic derivatives: A solution of 2 ml acetoneand 10 ml of ice water containing 0.89 g of cyanuric chloride is addedeach to (I) and (II).

    ______________________________________                                        (I) Acid derivative:                                                          4-aminobutyric acid (pK = 4.6)                                                                          0.48 g                                              trimethylamine            2.2 ml                                              sodium hydroxide, 8.6 mmol                                                                              8 ml                                                (II) Basic dervative:                                                         4-(2-aminoethyl)morpholine (pK = 6.0)                                                                   0.563 g                                             trimethylamine            2.2 ml                                              sodium hydroxide, 4.2 mmol                                                                              8 ml                                                ______________________________________                                    

Final concentration of derivatives in each solution is 0.195 M.Preparation of agarose membranes with immobilized pH determining groups:40 ml of 2% agarose solutions are prepared for casting two PIEFmembranes.

(A) 0.4 g of agarose (Type VIII-Sigma A-4905) are dissolved in 16 ml ofdeionized water and heated to 90° C. for half an hour. This solutionwill be derivatized with the aminobutyric acid buffer (I).

(B) An agarose solution as in (A) is prepared for reaction with themorpholine buffer (II).

(C) Both solutions are heated and kept at 50° C. for 40 minutes.

(D) 4 ml of an ice cold aqueous solution of dichlorotriazineaminobutyric acid (pK=4.6) are added slowly (3-4 minutes) to one of thesolutions of (C).

(D) 4 ml of an ice cold aqueous solution of dichlorotriazine aminoethylmorpholine (pK=6.2) are added slowly (3-4 minutes) to the other solutionof (C).

(F) Calculated amounts of solutions (D) and (E) are mixed at 90° C. forone hour to get the desired pH.

(G) The solution of (F) is poured into the PIEF mold (90 cm in diameterand 3 mm deep) at 90° C., covered with a glass plate and left to gel.

The obtained gel membranes are designed to clean the protein Eglin witha pI of 5.50. Therefore, the membrane on the anodic side was made with aBR concentration to give a buffered pH of 5.45, while the cathodicmembrane was buffered to 5.80. These membranes are then placed in thePIEF device according to FIG. 1. The details of each duplicate run(batches 1 and 2) are given in Tables 1 and 2. Impurities of the Elginsamples are separated within 0.05 pH units. 91% of protein recovery isachieved. Cleaning of impurities improves as a function of time.Cleaning for a longer period of time would get rid of the remainingimpurities.

EXAMPLE 2

The procedure of Example 1 is repeated, but the acid and the basicbuffered agarose products are prepared individually by heating them insolution, cooling the sol to form a gel, and then the gel is cleaned ofunreacted monomers and reagents (e.g. by dialysis and/orelectrophoresis), and the basic agarose products is then titsated in themelt form with the acid agarose product to give the desired pH of 5.5.The results in cleaning Eglin are even better than in Example 1.

EXAMPLE 3

In another variation of preparing the acid/basic triazine derivatives onthe agarose matrix, the 2% agarose gel is washed with acetone to replacewater; the agarose gel is then reacted with cyanuric chloride in acetoneat 0° C. for 30 minutes, and washed with acetone to take out theunreacted cyanuric chloride. The agarose dichloro triazine derivative isthen immersed in an acetone solution of the amino butyric acid oraminoethyl morpholine for 10 hours, the gel is then removed from thesolution and washed. PIEF membranes are made by dissolving thederivatives at 50° C. in water, titrating the basic derivative with theacid one to get gels of pH 5.8 and 5.65, and casting them intomembranes. These membranes cleaned Eglin on both sides of the anodic andcathodic bonds.

EXAMPLE 4

In another variation of making agarose derivatives, the dichlorotriazine amino butyric acid and aminoethyl morpholine each are dissolvedin acetone or an acetone-water solution at 0° to 4° C., proton acceptorsare added, such as diisopropylethyl amine or N,N-dimethylaniline, andreacted with agarose gel swollen with acetone for 10 to 72 hours,drained, washed and then used as in Example 3.

EXAMPLE 5

The agarose derivatives of Example 4 are titsated one against the otherwith a pH gradient mixer according to FIG. 2. In this case, however,dichloro triazinyl glycine was used instead of the aminobutyric acidderivative. The resultant gel (in a cassette) has a linear immobilizedgradient from pH 5 to pH 6 over a 10 cm length.

The single steps can be defined as follows:

prepare agarose-cycl-gly and

agarose-cycl-aem derivatives;

Melt the gels and prepare solutions of:

agarose-cyclogly (light))

agarose-cycl-aem+glycerol (heavy);

Create a pH gradient by the gradient former of FIG. 2. Solidify thegradient pH gel at 25° to 30° C. (cycl=cyanuric chloride; gly=glycine;aem=aminoethyl morpholine).

EXAMPLE 6

A. Preparation of 100 ml of a 2% agarose gel with 5 to 6 meq/kg(milliequivalents per kilogram) of 2% swollen gel bound aminoethylmorpholine (aem).

(1) Preparation of dichlorotriazinyl-aminoethyl morpholine:

(a) 1.6 g (8.7 mmol) of fresh crystallized cyanuric chloride aredissolved in 24 ml of acetone cooled to 0° C.

(b) To 0.35 g (8.7 mmol) of sodium hydroxide dissolved in 16 ml ofwater, cooled to 0° C., 1.36 ml (10.4 mmol) of4-(2-aminoethyl)-morpholine (aem) are added.

Solution (b) is then slowly added to solution (a) while stirring andcooling to 0° to 4° C. Stirring is continued at this temperature for 30minutes.

(2) Preparation of the agarose-cyanuric chloride-aem product:

2 g of agarose (Sigma A-4905) are placed into a 250 ml three-neckedflask. After adding 60 ml of aleionized water, the mixture is stirred atroom temperature for 10 minutes. The temperature is then raised to 90°C. under stirring until dissolution of the agarose. The temperature isthen decreased to 50° to 55° C. and the gel is equilibrated at thistemperature. The pH is adjusted to a value of 9 to 10 with 0.1N sodiumhydroxide solution. 20 ml of solution (1) is added, and the pH isadjusted to 9 to 10 with 0.1N sodium hydroxide solution. The mixture isstirred at 50° C. for 1 hour, maintaining the pH at 9 to 10. Then anadditional 20 ml of solution (1) is added, keeping the pH at 9 to 10.Stirring is continued for an additional one hour at 50° C., then 1.1 ml(8.5 mmol) of aem in a mixture of 6 ml of acetone and 2 ml of water isadded.

The temperature is then raised to 85° to 90° C. and the reaction mixtureis stirred at this temperature for one hour. The solution is then pouredinto a glass mold which is 20 cm long, 10 cm wide and 5 mm deep. Themold is left at room temperature for 3 hours and then stored at 4° C.for 12 hours.

The mold is then cut into pieces (1×1) and then washed as follows: 2times with 11 of an acetone/water mixture (80/20), 2 times with 1 l ofdeionized water, 2 times with 1 l of water acidified to pH 4, 2 timeswith 1 l of deionized water, 3 times with 1 l of water of pH 1 l (1 houreach), and then with aleionized water until the pH of water is achieved.

B. Preparation of of 100 ml of a 2% agarose gel with 6 to 7 meq/kg boundglycine.

(3) Preparation of dichlorotriazinyl-glycine:

(a) 1.6 g (8.7 mmol) of cyanuric chloride are dissolved in 20 ml ofacetone and 4 ml of cold deionized water; the solution is then cooled to0° C.

(b) 0.78 g (10.4 mmol) of glycine and 0.42 g (10.4 mmol) of sodiumhydroxide are dissolved in 10 ml of cold deionized water; the solutionis then cooled to 0° to 4° C.

(c) 0.34 g (8.7 mmol) of sodium hydroxide are disolved in 6 ml ofaleionized water. Solution (b) is then slowly added to solution (a) at0° C.; the pH is adjusted to 6.7 by adding solution (c). The obtainedreaction solution is stirred for 20 minutes at 0° to 4° C.

(4) Preparation of the agarose-cyanuric chloride-glycine product:

Procedure (2) is repeated, by using solution (3) instead of solution (1)and adding 0.64 g of glycine in 4 ml of aleionized water in the laststep.

The following washing procedure is used: 2 times with 1 l of deionizedwater, 2 times with 1 l of water of pH 4, 2 times with 1 l of deionizedwater, 2 times of 1 l of water of pH 8, 2 times of 1 l of deionizedwater, 3 times with 1 l of pH 3 (1 hour each), and finally withdeionized water until the pH of the water is achieved (3 to 4 l).

A pH gradient gel of between pH 5 to 6 is made from the abovederivatives using the gradient gel mixer in the following way: (1) Theaem/agarose derivative is titrated to a pH of 4.9 with theglycine/agarose derivative to give the "acidic solution", and to pH 6.1to give the "basic solution". (2) Using the gradient maker, the basicsolution is titrated continuously with the acidic solution to give therequired pH gradient.

Mixtures of protein markers (pI 5.1, 5.4, and 5.9) are separated on thisIPG gel. The results show a good separation and an almost linear pHgradient. In addition the individual markers are resolved into theircomponents showing a resolution of 0.02 pH units. These same mixtures ofprotein markers are resolved into patterns with the same band positionsshowing the uniformity along the length of the gel. The same approachcan be used to make gels in a range of e.g. pH 2 to 10 using theappropriate buffer derivatives, as shown in the following Example 7.

EXAMPLE 7

The necessary dichlorotriazines for each pH range are synthesized andused to prepare the agarose derivatives for the buffers and titrants.The reaction of the agarose with the the dichlorotriazines is conductedin aqueous solutions at the required concentrations (for details cf.Example 6 and the other foregoing examples) to give an acidic or basiccapacity between 4 to 20 meq/kg of a 2% wet gel. The agarose derivativesare then used as either buffers or titrants, depending on the IPG gelbeing made.

The following reactive dichlorotriazine derivatives are used:

    ______________________________________                                                             Agarose gel capacity                                                     pK.sup.1)                                                                          (meq/kg of 2% wet gel)                                   ______________________________________                                        Basic derivatives                                                             R--NH--(CH.sub.2).sub.2 --N(CH.sub.2).sub.4 O.sup.2)                                            6.2    6.7                                                  R--NH--(CH.sub.2).sub.3 --N(CH.sub.2).sub.4 O.sup.3)                                            7.0    4.3                                                  R--NH--(CH.sub.2).sub.2 --N--H(CH.sub.3).sub.2.sup.4)                                           8.5    7.5                                                  R--NH--(CH.sub.2).sub.3 --N--H(CH.sub.3).sub.2.sup.5)                                           9.3    14                                                   Acidic derivatives                                                            R--NH--CH.sub.2 COOH.sup.6)                                                                     3.6    9.3                                                  R--NH--(CH.sub.2).sub.3 --COOH.sup.7)                                                           4.6    12.9                                                 ______________________________________                                         .sup.1) These values are taken from polyacrylamideimmombilines; for the       triazinyl derivatives bound to agarose the pK's may be different.             .sup.2) 4(2-Aminoethyl)-morpholine derivative (AEM)                           .sup.3) 4(2-Aminopropyl)-morpholine derivative (APM)                          .sup.4) N,NDimethylethylenediamine derivative (DMEDA)                         .sup.5) N,NDimethylpropylenediamine derivative (DMPDA)                        .sup.6) Glycine derivative (GLY)                                              .sup.7) 4Aminobutyric acid derivative (ABA)                              

The agarose derivatives are mixed at a temperature at which they aremelted (usually about 60° C.), while the IPG solid gel is used inanalytical IEF (isoelectric focusing) at 10° C. Since the pH of the gelis temperature-dependent, this dependency has to be considered in orderto predict the gel's pH at 10° C., based on the known titrated pH at 60°C. Thus, the operational steps are:

The agarose buffer is titrated with the agarose to the basic extreme ofthe gel.

The pH vs. temperature dependency is determined and the pH at 10° C. iscalculated.

The pH at 60° C. is readjusted to get the desired pH at 10° C.

A similar operational approach is conducted with the acidic pH extremeof the IPG.

To generate a linear pH gradient, a constant buffer capacity over thegradient is required. To achieve this constant buffer capacity, theconcentration of the buffering species is adjusted so that theconcentration of buffer increases, going to the pH extreme farthest fromthe pK of the buffer. In this case it is achieved by the addition ofglycerol to the appropriate buffer/titrant solution.

pH-range: 4 to 5

In this range the ABA-derivative (pK 4.7) is used as the buffer andtitrated with the DMPDA-derivative (pK 9.3). The pH vs. temperaturecurve for the acidic and basic extremes showed the need to titrate a 60°C. solution at pH 4.7 with a solution at pH 5.5 to get an IPG gel of pH4.0 to 5.0. The first gel, however is titrated to pH 4.4 and 5.0 at 60°C. to give a pH range of 4.0 to 4.7 at 10° C.

The gel shows a good separation of protein markers 4.2 and 4.6.

pH-range: 5 to 6 (cf. Example 6)

pH-range: 6 to 7

In this range the buffer is the APM-derivative and the titrant is theGLY-derivative. The pH vs. temperature curve fixes the acidic side at pH5.2 and the basic side at pH 6.5 at 60° C. for achieving a functioninggel in the range of pH 6 to 7 at 10° C.

pH-range: 7 to 8

An IPG gel in this range is made with the DMEDA-derivative as the bufferand the GLY-derivative as the titrant. A good separation between themarkers 7.4 and 7.2 is achieved, although the pK of the buffer (8.5') isoutside of the IPG range of 7 to 8.

                                      TABLE 1                                     __________________________________________________________________________    % Protein Recovery = 91% batch 1                                              __________________________________________________________________________    Time (min)                                                                            1   7       29  32      72  83                                        Volt    300 825     1072                                                                              1067    1056                                                                              1059                                      mA      30  30      28  28      28  28                                        Watt    9   25      30  30      30  30                                        pH      7.3 5.8     5.2 5.3     5.5 5.5                                       μS   9.5 1.8     1.4 1.4     1.4 1.4                                       __________________________________________________________________________    EXPERIMENTAL CONDITIONS                                                       Cathodic Reservoir                                                                      Membranes                                                                            Center channel                                                                         Membrane 2                                                                            Anoid reservoir                             __________________________________________________________________________    1 mM NaOH pH: appx. 5.8                                                                        2 mg/ml EGLIN                                                                          pH: appx. 5.45                                                                        1 mM Acetic Acid                            125 ml           100 ml           125 ml                                      __________________________________________________________________________

                                      TABLE 2                                     __________________________________________________________________________    % Protein Recovery = 91% batch 2                                              __________________________________________________________________________    Time (min)                                                                            1   6       7   30      68  80                                        Volt    303 565     1000                                                                              1052    1027                                                                              1030                                      mA      30  30      29  29      29  29                                        Watt    9   17      30  30      30  30                                        pH      7.4 6.2     5.8 5.5     5.5 5.5                                       μS   12  2.5     1.5 1.4     1.4 1.4                                       __________________________________________________________________________    EXPERIMENTAL CONDITIONS                                                       Cathodic Reservoir                                                                      Membranes                                                                            Center channel                                                                         Membrane 2                                                                            Anodic reservoir                            __________________________________________________________________________    1 mM NaOH pH: appx. 5.8                                                                        2 mg/ml EGLIN                                                                          pH: appx. 5.5                                                                         1 mM Acetic Acid                            125 ml           80 ml            125 ml                                      __________________________________________________________________________

We claim:
 1. A gradient mixer for the preparation of immobilizedpH-gradient gels which comprises(A) a first temperature-controlledcirculating air oven (7) comprising(a) a glass syringe (4) fitted with aplunger and an output flexible tube, and being adapted to receive abasic or an acidic gel; (b) a glass syringe (5) fitted with a plungerand adapted to receive a basic or an acidic gel, provided that ifsyringe (4) contains a basic gel, then syringe (5) contains an acidicgel, and vice versa; (c) a magnetic mixer (6) adapted to mix said basicand acidic gels; (d) a load screw (3) driven by a motor (1) at a speeddetermined by a motor speed controller (2) having an upper limit switch(15) and a stop point (low limit) switch (17), load screw (3) beingadapted to depress the respective plungers of the syringes (4) and (5)and eject said basic and acidic gels from the respective syringes (4)and (5); (e) a rotatable three way valve (14) connecting syringes (4)and (5); (f) an air jet gun (10) adapted to heat said oven (7); and (g)a temperature controller and indicator (12) adapted to control thetemperature of said oven (7); (B) a second temperature-controlledcirculating air oven (8) comprising(a) a cassette (9) adapted to receivemolten gel directly from syringe (4) via said output flexible tube, andto receive a mixed solution of said basic and acidic gels via saidsyringe (5), said valve (14), syringe (4) and said output flexible tube,the volume of said mixed solution being controlled by said upper limitswitch (15) and said stop point (low limit) switch (17); (b) an air jetgun (11) adapted to heat said oven (8); and (c) a temperature controllerand indicator (13) adapted to control the temperature of said oven (8);and (C) an electric switching board (18) adapted to monitor saidgradient mixer.